Abstract
To determine the effect of various stereoisomers of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE) on translesion bypass by human immunodeficiency virus-1 reverse transcriptase and its alpha-helix H mutants, six 33-mer templates were constructed bearing site- and stereospecific adducts. This in vitro model system was chosen to understand the structure-function relationships between the polymerase and damaged DNA during replication. Comparison of the replication pattern between wild type human immunodeficiency virus-1 reverse transcriptase and its mutants, using primers which were 3' to the lesion, revealed essentially similar patterns. While these primers terminated with all three of the C10R and two of the C10S BPDE-adducted templates 1 base 5' and 1 base 3' to the damaged site respectively, (+)-anti-trans-(C10S) BPDE-adducted DNA alone permitted the formation of full-length products. Utilization of a primer with its 3'-hydroxyl 1 base beyond the lesion resulted in full-length products with all the C10S BPDE-adducted templates and the (-)-syn-trans-(C10R)-BPDE-adducted template, following replication with either the wild type or mutant enzymes. However, the other two C10R BPDE-adducted templates failed to allow any primer extension, even with the wild type enzyme. Although T.P depletion studies further confirmed the differential primer extension abilities using the C10R and C10S adducted templates, their binding affinities were similar, yet distinct from the unadducted template.
Highlights
Benzo[a]pyrene is a ubiquitous by-product of incomplete combustion and is one of the most potent carcinogens known [1,2,3,4]
Structural data on double-stranded oligonucleotides adducted with BPDE at N2 of guanine revealed that the pyrenyl moiety of (ϩ)-anti-trans-isomers with the S configuration at C10 is directed toward the 5Ј end of the modified strand
Insight into the precise nature of the interactions between the different stereospecific BPDE-adducted templates and various polymerases has involved numerous in vitro studies, where generally, termination occurs at or one base prior to the site of the adduct [26, 27, 32,33,34]. Translesion synthesis with these bulky adducted templates has to date been observed with relatively few polymerases [20, 23]
Summary
Benzo[a]pyrene is a ubiquitous by-product of incomplete combustion and is one of the most potent carcinogens known [1,2,3,4]. HIV-1 reverse transcriptase (HIV-1 RT) is a hypermutable enzyme that catalyzes the addition of approximately 20,000 nucleotides with extremely low fidelity during replication, resulting in mutations at a frequency of 1/1700 nucleotides inserted [35,36,37] Both the crystal structure and the co-crystal structure with duplex DNA are known and numerous site-specific mutants that were designed with structure-function aspects in mind are readily available (38 – 41). Because the amino acid residues 262 and 266 are in contact with the duplex region in the vicinity of the DNA minor groove, relatively close to the 3Ј terminus of the primer, these two mutants were utilized in the present study These polycyclic aromatic hydrocarbon adducts are not likely to be encountered by HIV-1 RT in vivo, they serve as an ideal model for examining the effect of bulky adducts on DNA processing by polymerases
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